Vibrio cholerae causes the frequently fatal epidemic diarrheal disease cholera. The expression of its two primary virulence factors, toxin-coregulated pilus and cholera toxin, occurs via a transcriptional cascade involving several activator proteins and serves as a paradigm for the regulation of bacterial virulence. AphA and AphB initiate the expression of the cascade by an as yet not understood synergistic interaction at the tcpPH promoter. AphA is a member of a new and uncharacterized regulator family and AphB is a LysR-type activator, one of the largest transcriptional regulatory families. Once expressed, cooperation between the homologous transmembrane regulators TcpP/TcpH and ToxR/ToxS activates the toxT promoter. ToxT, an AraC-type regulator, then directly activates the promoters of the primary virulence factors. Transcriptional activation at these various promoters occurs only in response to certain environmental stimuli. Such regulation is widespread among bacterial pathogens and allows productive infections to be mounted only in the appropriate biological niches. The long term goals of the work in this proposal are to understand the molecular basis for this regulation by environmental stimuli, so as to facilitate the development of better strategies to prevent and cure bacterial diseases. Achieving these goals requires an understanding of how the activators themselves function to initiate gene expression and, ultimately, how they are influenced by particular environmental stimuli. Through a collaborative effort of laboratories with expertise in structural biology, virulence gene regulation and pathogenesis, this proposal aims to explore the structure/function relationships of the three cytoplasmic vinflence gene regulator proteins in V. cholerae, AphA, AphB and ToxT, at their cognate promoters. Specifically, we propose to obtain high resolution structures of (1) AphA; (2) AphB; and (3) ToxT in the absence and presence of their binding sites. In combination with ongoing mutational studies, the proposed work will significantly increase our understanding of how these proteins activate virulence gene expression, will serve as models for these regulatory protein family members in other bacterial pathogens, and will advance efforts to identify molecules that may function as novel therapeutics.